The invention concerns methods and equipment for the mass spectrometric measurement of a large number of genotyping profiles, each formed by many SNPs (single nucleotide polymorphisms) in a DNA sample, and for the associated sample preparation.
There is a growing demand for easy, fast and economical generation of specified genotyping profiles, each involving the determination of many SNPs in a DNA sample, partially up to several tens or even to several hundreds of SNPs. Such a profile can, for instance, identify a person or an animal, to provide evidence for the responsibility for a crime, or to exclude fraudulent substitutions (for example, in the case of race horses, cattle, pedigree dogs or pigeons). With about 50 SNPs a person can be uniquely identified from a tiny sample of DNA. This kind of profile can also provide evidence of ancestry, as proof of paternity, or as proof of the pedigree of cattle.
A particularly important use of genotype profiles, however, is the preventive detection of predispositions to disease, for instance a tendency towards thrombosis, or for the purposes of individualized medication (the xe2x80x9cpersonal pillxe2x80x9d). It is to be expected that the measurement of such genotype profiles with high analytic reliability will play an important role in the medicine of the future. The analytic reliability required for this can, to date, only be guaranteed by mass spectrometry.
Thus the field of this invention is a method for the detection of a large number of specific mutational changes in the genomic DNA in the course of a single, easy analysis process, where the mutation sites themselves are known and are specified for the genotyping task. As far as the mutational sequence changes are concerned, particular attention is paid to the simple exchange of bases (xe2x80x9cpoint mutationxe2x80x9d), which has become known recently under the abbreviation xe2x80x9cSNPxe2x80x9d (single nucleotide polymorphism). For human beings it is believed that at least three million SNPs occur frequently, which characterize many of the individual differences between people and control the individual genetic predisposition.
Usually a xe2x80x9cwild typexe2x80x9d is defined for a genome, and this xe2x80x9cwild typexe2x80x9d is considered to be free from mutations. Bearing in mind the frequency of mutations, for instance of SNPs, and the equal validity of the mutated type (mutants) and of the wild type, the definition of the wild type is arbitrary, or at least a matter of chance.
All the mutations of DNA considered here result in a difference in the mass of the segment of DNA containing this mutation as compared with the mass of the corresponding section from the wild type. This means that precise determination of the mass of a segment of DNA can be used to determine a mutation.
Mass spectrometry is an extremely powerful method for measuring the masses of biomolecules. The mass of the ions can be analyzed by mass spectrometry, for instance in time-of-flight mass spectrometers using ionization by matrix assisted laser desorption (MALDI). But ionization by electrospray ionization (ESI) can also be used, although usually in association with mass spectrometers of a different type.
Polymerase chain reactions (PCR) can be used to manufacture selected double-strand DNA products with a minimum length of about 40 base pairs by application of a pair of xe2x80x9cselection primersxe2x80x9d, single-strand oligonucleotides with a length of about 20 bases, in a known manner. The mutation site must be included by corresponding selection of the sequence of the two selection primers.
The obvious process of using mass spectrometry is simply to measure the mass of DNA products multiplied by PCR and so to determine the mutations. This process has been found almost impossible to implement, because accurate measurement of the masses of DNA products with lengths of more than 40 base pairs has proved impossible in practice. The reasons for this are given below.
Methods have therefore been sought that yield shorter DNA fragments. For this purpose, the process of limited, mutation-dependent primer extension was first developed, which generates extended primers having a length of about 15 or 25 nucleotides, from whose mass the type of mutation can be determined more effectively. Other improvements consist in the removal of a large piece of this extended primer, for instance by enzymatic digestion of a piece; the details of this will not be described any more closely here.
The invention of photo-cleavable linkers brought further progress. The linkers are integrated into the extension primers, and bridge one nucleotide without disturbing either the hybridization or the enzymatic extension, and can be cleaved by means of UV light following preparation of the sample. This allows small fragments with lengths of only 4, 5 or 6 nucleotides to be obtained, and these can be very effectively ionized using matrix assisted laser desorption and ionization (MALDI).
The MALDI preparation and measurement procedure consists in first embedding the analyte molecules on a sample carrier in a solid, UV-absorbing matrix, usually an organic acid. The sample carrier is inserted into the ion source of a mass spectrometer. A short laser pulse, about three nanoseconds in duration, is used to vaporize the matrix into the vacuum; during this process the analyte molecules are transported into the gaseous phase largely, though unfortunately not completely, unfragmented. The molecules of analyte are ionized by proton transfer as a result of impacts with matrix ions that are created at the same time. An applied voltage accelerates the ions into a field-free flight tube. Because of their different masses, the ions are accelerated in the ion source to different velocities. Smaller ions reach the detector earlier than larger ions. The measured flight time is used to calculate the masses of the ions.
MALDI is particularly suitable for the analysis of peptides and proteins. The analysis of nucleic acid chains is more difficult, and is only adequately effective for short-chain nucleic acids. The reason for this is that only a single proton needs to be captured to ionize peptides or proteins to form a positive ion, whereas nucleic acids form a poly-anion with multiple negative charges at the sugar phosphate backbone (one negative charge for each nucleotide), and the ionization process to form a positive ion is significantly less efficient because it needs the transfer of a multitude of protons from a multitude of matrix ions. It is only of adequate efficiency for very short chains, such as for the cleavage products of the extended primers, as can be created with the aid of photo-cleavable linkers.
It is a well-known and favorable embodiment for the analysis of genotyping profiles of DNA samples to use chips on which sufficient numbers of different types of extension primers are bonded in separate locations as probes for the selected SNPs of the genotyping profile. In association with the use of cleavable primers, as discussed above, this therefore provides a powerful tool for the mass spectrometric analysis of genotyping profiles.
The invention consists of providing a multitude of chips held simultaneously in a combining structure, as well for the sample preparation in a matching multitude of processing wells and as for the joint mass spectrometric analysis for the determination of numerous genotyping profiles. For this purpose the chips are held by the combining structure in such a way that they can be fed as a rigid unit to the multitude of wells with DNA samples as well as to the mass spectrometric analysis. The multitude of wells can, for instance, be a microtitre plate.
The combining structure can be a flat plate containing the chips as parts of its surface and which is pressed closely onto the processing wells, such as the wells of a microtitre plate, so that it comes into contact with the liquid inside the wells when the structure with the processing wells is inverted.
The linking structure is, however, preferably a plate on which the chips sit rigidly on small stems or pillars, so that when this chip carrier plate is turned over the chips can be immersed simultaneously in the wells, for instance in the wells of a microtitre plate, at the same time closing the wells.
Both types of carrier plates mayxe2x80x94as they stand, or with an additional framexe2x80x94be inserted into the mass spectrometer. An electrically conducting top frame is particularly necessary for the chip carrier plate in which the chips are on stems, so that the electrical potential gaps between the chips are filled and the chips may be electrically contacted for use in the mass spectrometer. It is favorable for the acceleration of the ions created on the chips by pulsed laser desorption to originate from a wide and even plane with identical electrical potential throughout the plane.
In order for the chips to be properly positioned in the top frame for mass spectrometric analysis and accurately located in one plane, it is expedient to make the stems of the chips elastic, and for the chips to be precisely shaped so that they fit positively into the corresponding negative shapes in the top frame. The top frame will then provide very precise adjustment to the chips, both laterally and in reference to the plane.
Each of the chips carries the oligonucleotide probes that have to be provided with the chip for processing and prepared for mass spectrometric analysis. These probes are bonded to the surface in a large number of compartments. One known processing method is limited mutation-dependent primer extension, in which the oligonucleotides are extended enzymatically by precisely one nucleotide after the deposition of template strands that carry the mutation. The nucleotide used for the extension thus carries the information about the mutation. It is very expedient for the oligonucleotides to carry a cleavable linker not far from the 3xe2x80x2 end that can be cleave after the processing, and supplies uniformly short fragments, for instance only 5 nucleotides in length, which carry the mutation information and can easily be measured by mass spectrometry.
In order to detect and compensate for the residual differences in the height of the chips within the potential plane, it is expedient to have reference signals with precisely known mass in the spectrum of each compartment. These are helpful for determination of the mass, because the flight times of MALDI ions are shifted as the separation from the nearest acceleration electrode varies. These mass references can easily be added to the individual compartments when the chips are manufactured. They consist of reference primers with terminating ends, and also having cleavable linkers. These are not extended, but supply cleavage products with precisely known masses. It appears that even one such reference primer in each compartment is sufficient, although two reference primers for each compartment, located as close as possible to the ends of the range of masses, are better.
A further idea of the invention is to provide a ready made application kit for the determination of a genotyping profile, containing at least the chip carrier plates with chips, each having the oligonucleotide probes for complete genotyping and the necessary primer pairs for multiplex PCR amplification of the DNA sample for manufacture of the template.
It is also possible for a package to additionally contain the NTPs for the amplifying PCR process, the cleaning media, the mixture of terminating ddNTPs and the data relating to the masses of the cleave segments to be expected in each compartment on a computer readable medium. It is even possible to include an executable computer program that calculates the medical, breeding or other relevant results according to the latest state of knowledge from the measured genotyping profile. It is furthermore possible to include the most favorable polymerases for the primary PCR amplification of the templates and for the primer extension and purified matrix substances for the MALDI ionization.
For urgent analyses it can be appropriate to have chip carrier plates with smaller numbers of chips available. This will shorten the measurement time in the mass spectrometer. Thus a carrier plate with 12 chips, at a measurement time of 1 second per compartment, can be measured in half-an-hour, whereas the measurement of a carrier plate with 96 chips takes four hours, and in any event is only possible with the most modem mass spectrometers. The time for preparation of the samples, however, is only shortened slightly.